We previously evaluated two different paramyxoviruses as vaccine vectors for human intranasal immunization. One was human parainfluenza virus type 3 (HPIV3). HPIV3 was of interest because we presently are developing attenuated versions of HPIV1, 2, and 3 as pediatric vaccines, and it was useful in addition to evaluate their potential as vaccine vectors against heterologous agents. The other paramyxovirus that we evaluated is the avian paramyxovirus (APMV) Newcastle disease virus (NDV). NDV is highly attenuated in primates due to a host range restriction. In each case, we used reverse genetics to insert (into HPIV3 or NDV) an additional gene expressing the foreign antigen of interest. We made HPIV3 vectors expressing the spike glycoprotein of Severe Acute Respiratory Syndrome Coronavirus (SARS) or the Ebola virus glycoprotein GP. These were immunogenic and protective in challenge studies in rodents and non-human primates. We also made NDV vectors expressing SARS S, Ebola virus GP, and the hemagglutinin HA or neuraminidase NA glycoprotein of highly pathogenic avian influenza virus. In each case, the experimental vaccines were immunogenic and, when tested, protective against challenge. With the HPIV3 vectors, a single dose typically was used, whereas the NDV vectors are much more highly attenuated and usually were given in two doses. At the present time, we are further evaluating two versions of HPIV3-vectored vaccines that will be reported on next year. NDV represents serotype 1 of the APMVs. There are 8 other established APMV serotypes, 2 to 9. NDV has been extensively characterized because of its importance in poultry. In contrast, relatively little was known about the other serotypes. Working with collaborators at the University of Maryland at College Park, we previously initiated antigenic and sequence analysis of these as a prelude to their development as potential vectors. We have determined complete sequences for at least one, and in most cases several, strains from each serotype, an analysis that is ongoing as we obtain new isolates worldwide. We have characterized diversity, confirming the serotype distinctions as well as identify antigenic heterogeneity within some subgroups. We also continue to evaluate these viruses for infectivity, replication, and pathogenesis in domestic poultry and rodents. In general, this has shown that these viruses usually are non-pathogenic in poultry, which is important for agricultural safety. They also are attenuated in rodents. At the present time, selected viruses are being evaluated in non-human primates, which will be reported on next year. We have been investigating the effects of manipulating specific features of the APMVs that, based on published work with other enveloped viruses, may modulate replication and virulence and thus could provide modulation of vaccine properties. Two examples will be reported here: namely, manipulation of glycosylation sites in the F protein, and manipulation of the cleavage site in the F protein. We evaluated the role of N-linked glycosylation in the F protein of NDV by using reverse genetics to delete potential acceptor sites in complete virus. This was done with the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1-6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, which interact during fusion. Each N-glycosylation site was eliminated individually by substituting asparagine (N) with glutamine (Q), and a double mutant (Ng2+5) involving the two HR domains also was made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. Mutations at each of the other 5 sites resulted in increased F protein electrophoretic mobility, suggesting that each site normally is occupied. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multi-cycle growth in vitro. These three single mutations also were the most attenuating in eggs and 1-day-old chicks, and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was somewhat more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral and CD4+ T cell responses. However, these viruses remained substantially less virulent than velogenic strains of NDV. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology, and suggest that the N-glycans in HR1 and HR2 coordinately down-regulate viral fusion and virulence. We are presently investigating whether this approach can be used with highly attenuated APMV strains to increase the level of replication in vitro and in vivo. We also investigated the effects of mutations in the cleavage site of the F protein of APMV-7. In related viruses, the ability of the F protein to be cleaved is a major determinant of replication efficiency, tissue tropism, and virulence. This study employed an APMV-7 reverse genetics system that we developed. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR&#8595;FA). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. This was unusual, because typically monobasic cleavage sites depend on added protease for cleavage in cell culture. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent NDV isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR&#8595;FI) or (RRKKR&#8595;FI), named Fcs-4B or Fcs-5B, respectively. Each of these sites contains the preferred furin cleavage sequence RX(K/R)R&#8595;. A third APMV-7 mutant (LRSKR&#8595;FI) that was designed to have 3 basic residues that conformed to the furin site could not be recovered, implying that additional cleavage site residues are important. In cell culture, the Fcs-5B virus caused protease-independent syncytia, formed plaques, and grew to 10-fold higher titers compared to the parent virus, whereas the Fcs-4B virus did not form syncytia or plaques and did not have improved growth. This indicated the importance of the single additional basic residue (K) at position -3. Parental APMV-7 had a mean embryo death time of >168 h and an intracerebral pathogenicity index of 0, indicating that it is avirulent in chickens. These values were unchanged in the two cleavage site mutants. In addition, parental APMV-7 was limited in tropism in 1-day-old and 2-week-old chickens to the upper respiratory tract, and this was unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation, plaque formation, and increased replication in vitro, but did not alter replication, tropism, or virulence in chickens. In conclusion, we identified two means of modulating the replication, immunogenicity, and virulence of representative APMVs. This likely will be important in developing an optimal APMV vaccine vector.